Method for producing biomass using hydrogen-oxidizing bacteria

This application is the US National Stage Application of International Patent Application No. PCT/EP2021/060880, filed Apr. 26, 2021, which claims the benefit of the priority of Great Britain Patent Application No. 2006071.1, filed Apr. 24, 2020, the disclosures of which are incorporated herein by reference in their entireties.

The present invention provides a method for producing a biomass comprising at least 65% protein from hydrogen-oxidising microorganisms using one or more input streams comprising one or more gaseous substrates.

Production of protein as a food source in a more sustainable fashion is needed to reduce resource use and greenhouse gas (GHG) emissions. A growing global population puts increasing pressure on the availability of resources and the environment. Animal products such as meat, fish, milk and eggs are important dietary sources of protein, but livestock use a large area of agricultural land, energy and water. This is mainly due to the fact that animal feed consists of large quantities of plants specifically grown for this purpose. An alternative source of the protein component of animal feed or even for direct human consumption is microbial protein, which can be tailored in composition to suit specific nutritional needs. Microbial protein is considered to be a highly sustainable source of protein, due to the efficiency of land, energy, and water usage in its production.

Many industrial processes generate off-gas streams that comprise carbon dioxide among other gas components. Examples are the production of energy through combustion, lime, fertilizers, and cement, which are major sources of atmospheric carbon dioxide, as well as other GHG. Carbon oxides from industrial sources are primarily produced by the combustion of fossil fuels and/or chemicals and are classified as GHG due to their contribution to deleterious environmental conditions. Other industrial processes that include combustion of wastes are municipal solid waste, sewage sludge, plastic, tires, agricultural residues and the like, as well as coal- or gas-fired electricity plants.

Microbes require a source of carbon in order to live, grow and produce chemical products. Thus, carbon oxides derived from industrial gas effluent represent a potentially cheap, sustainable, and scalable means to obtain carbon for microbially-mediated production of food rich in protein, and a way to reduce the amount of carbon dioxide released directly into the atmosphere.

Therefore, it would be desirable to be able to convert gaseous feedstocks into a source of high-quality biomass. Accordingly, there is a need for an improved, simple, highly productive and economical process for the biological production of biomass useful as e.g. animal feed or direct human consumption.

Prior work is known relating to certain applications of chemoautotrophic microorganisms in the capture and conversion of carbon dioxide gas to fixed carbon. However, many of these approaches have suffered shortcomings that have limited the effectiveness, economic feasibility, practicality and commercial adoption of the described processes. In particular, achieving a maximal productivity rate, preferably by maintaining a high concentration of the microorganisms in the liquid phase of the bioreactor, combined with a high protein content in a continuous stable manner is a challenge without making the process economically unattractive.

U.S. Pat. No. 9,157,058B2 (WO2013090769A2) describes an apparatus and method for growth and maintenance of microorganisms and/or bioprocesses using one or more gases as electron donors, electron acceptors, carbon sources, or other nutrients, and for a bioprocess that converts hydrogen and carbon dioxide, or syngas, or producer gas into lipid products, bio-based oils, or other biochemical products. However, it does not disclose optimal conditions for growth and maintenance of the microorganisms nor does it disclose a process to optimize their protein production capacity.

U.S. Pat. No. 9,206,451B2 describes systems and methods for employing chemoautotrophic micro-organisms to capture carbon from industrial waste, but it does not disclose controlled optimal process conditions for growth and maintenance nor a preferred microorganism nor does it disclose a process to optimize their protein production capacity.

Patent application WO2018144965A1 describes microorganisms and bioprocesses that convert gaseous substrates, such as renewable hydrogen and waste carbon dioxide producer gas, or syngas into high-protein biomass. However, it does not disclose controlled optimal process conditions for growth and maintenance of the microorganisms nor does it disclose a process to optimize their protein production capacity.

Patent application WO2019010116A1 describes a method for producing multi-carbon compounds from simple gas feedstocks, such as carbon dioxide, hydrogen and oxygen, by cultivating a consortium of microbial cells specially selected for this purpose in an aqueous culture medium. However, it does not disclose controlled optimal process conditions for growth and maintenance of the microorganisms, nor does it disclose a process to optimize their protein production capacity.

T G Volova and V A Barashkov; in: “Characteristics of Proteins Synthesized by Hydrogen-Oxidizing Microorganisms”; Applied Biochemistry and Microbiology, 2010, describe a process wherein hydrogen-oxidizing bacteria are cultivated to generate a biomass with a 64 to 76% dry weight protein content, but the amounts of carbon dioxide, hydrogen and oxygen used are not disclosed nor does it disclose a process to optimize productivity.

Patent application US2010120104A1 discloses a multistep method for producing biomass by capturing carbon via obligate and/or facultative chemoautotrophic microorganisms, and/or cell extracts containing enzymes from chemoautotrophic microorganisms. A multitude of different electron donors and acceptors and microorganisms for use in the method are disclosed and no specific process parameters are described.

Patent application WO2011139804A2 discloses a method for producing biomass by capturing carbon with one carbon atom by oxyhydrogen microorganisms and a suitable bioreactor utilizing hydrogen and oxygen gas, wherein the volume of gas occupies at least about 2% of the total volume of the column in which the volume is positioned. No specific process parameters are described for optimization of protein production capacity.

Patent application WO2017165244A1 discloses a method for producing biomass by the capture and conversion of inorganic and/or organic molecules containing only one carbon atom by chemoautotrophic microorganisms. It is disclosed thatwas grown on Hand COin standard off-the-shelf lab-scale bioreactors to dry biomass densities above 40 g/liter over 6 days. It is also disclosed thatstrains DSM 531 and DSM 541 grown in liquid MSM media with an unspecified Knallgas mixture as the sole carbon and energy source accumulated over 70% and over 80% total protein by weight, respectively, for samples taken during the arithmetic growth phase. However, it is disclosed thatis grown under oxygen-limiting conditions, which is non-optimal for production of biomass with high protein concentrations at a high, industrial rate. Moreover, the disclosed system used for growingis a continuous-fed-batch system, wherein the specific growth rate is a function of gas transfer rates that are not disclosed. Therefore, WO2017165244A1 does not disclose that biomass can be produced at a high rate, wherein the biomass comprises a high protein content, nor are the amounts of carbon dioxide, hydrogen and oxygen used disclosed for obtaining a biomass with a high protein content and/or high biomass production rate.

Morinaga et al. in “Growth Characteristics and Cell Composition ofin Chemostat Culture”; Agric. Biol. Chem., 1977, describe conditions for culturing, but they do not disclose how to achieve the production of biomass at a rate greater than about 7.2 g/l/d and together with a protein content greater than 65% nor do they disclose a growth medium capable of supporting a high concentration of microorganisms. Thus, combining a high productivity with a high density of microorganisms is a challenge.

Thus, there remains a need to identify a set of chemoautotrophic microorganisms that can grow in novel or conventional, controllable, and scalable contained reaction vessels, and that produce protein and other nutritionally beneficial products in a stable and commercially viable manner with high rates of productivity. The growth and maintenance of these microorganisms are then managed by controlled optimal process conditions to fine tune the metabolic and physiological profiles of the microorganisms, ultimately resulting in a high production of a high-quality biomass with a high protein content, preferably by maintaining a high concentration of the microorganisms in the liquid phase of the bioreactor.

The present invention provides a commercially viable method for producing a high-quality biomass containing a high protein content with a high productivity. This is achieved by using an input stream comprising a gaseous carbon and energy source managed by controlled optimal process conditions, a nutrient supply managed by controlled optimal process conditions and a culture of chemoautotrophic hydrogen oxidising microorganisms. This solution reduces and optimises substrate limitations and enables higher oxygen utilisation, and thereby improved productivity, preferably in a manner which maintains safe operating conditions within the bioreactor, which may be achieved through mitigation of potentially explosive gas mixtures.

The object of the present invention is therefore to provide a method for producing a biomass comprising at least 65% protein of total biomass by dry weight from hydrogen-oxidising microorganisms using one or more input streams comprising one or more gaseous substrates, comprising hydrogen and/or oxygen and/or carbon dioxide, comprising contacting the microorganisms in a liquid phase with a nutrient composition comprising carbon and/or nitrogen and/or phosphorous comprising compounds and the gaseous substrates wherein the input stream and nutrient composition are controlled and wherein the biomass is produced at a rate of more than 10 g/l/d.

It is a further object to provide a method, wherein controlling the input stream comprises maintaining a molar ratio of dissolved hydrogen:oxygen:carbon dioxide of from 0 to 12.748:0 to 4.25:0 to 2.0 in the liquid phase within a distance of from 0 to 500 mm, preferably of from 0 to 100 mm, from a gaseous phase, which is in direct contact with the liquid phase.

It is yet a further object to provide a method of producing biomass from bacteria selected from the genussp.

It is yet a further object to provide a method of producing biomass from bacteria selected from the genussp.

In a further aspect, the invention provides a method of isolating the produced biomass and removing the nutrient composition, comprising downstream processing.

It is a further object to provide a method of isolating the produced biomass by removing the nutrient composition, comprising dewatering and/or drying the biomass such that the biomass comprises a water content of less than 5% by weight.

It is yet a further object to provide a biomass comprising protein comprising an amino acid content comprising a histidine content of from 0.9 to 4.8% of total biomass dry weight protein content, an isoleucine content of from 2.0 to 6.9% of total biomass dry weight protein content, a leucine content of from 3.8 to 12.0% of total biomass dry weight protein content, a lysine content of from 3.0 to 11.1% of total biomass dry weight protein content, a methionine content of from 1.1 to 5.4% of total biomass dry weight protein content, a phenylalanine content of from 1.7 to 8.5% of total biomass dry weight protein content, a threonine content of from 1.6 to 6.9% of total biomass dry weight protein content, a tryptophan content of from 0.4 to 3.9% of total biomass dry weight protein content, and a valine content of from 1.7 to 9.3% of total biomass dry weight protein content.

In a further aspect, the invention provides a biomass comprising a lipid content of from 2.3 to 18% of total biomass dry weight comprising a fatty acid content comprising a C16:0 palmitic acid content of from 23 to 60% of total biomass dry weight fatty acid content, a C16:1 palmitoleic acid content of from 3.8 to 22.3% of total biomass dry weight fatty acid content, and a C17:1 heptadecenoic acid content of from 23 to 60% of total biomass dry weight fatty acid content.

It is yet a further object to provide a use of the nutrient composition obtained by a method of isolating the produced biomass as a nutrient composition for producing biomass.

Another object of the present invention is to provide a use of the biomass, wherein the biomass is used to feed or provide nutrition to one or more organisms.

The following list comprises definitions of reference numerals as employed in the figures:

Biomass herein is understood to mean the total weight of microorganisms and their progeny, products and/or metabolites.

Bioreactor is understood herein as to be a system for maintaining and/or growing microorganisms comprising a gaseous phase, commonly called the headspace, and a liquid phase. The microorganisms are grown and maintained in the liquid phase.

Hydrogen-oxidising microorganisms are meant to be understood to comprise facultative chemoautotrophic bacteria that can use hydrogen as electron donor. The group of aerobic hydrogen-oxidizing bacteria, also known as Knallgas-bacteria, is physiologically defined and comprises bacteria from different taxonomic units. This group is defined by the ability to use gaseous hydrogen as electron donor with oxygen as electron acceptor and to fix carbon dioxide.

Input stream is herein understood to mean a supply of nutrients and/or energy for growth and/or maintenance of microorganisms comprising a liquid phase and/or a gaseous phase.

Liquid phase herein is understood to mean a volume comprising liquid material. In the liquid phase microorganisms are commonly grown and maintained. Biomass mostly subsists within the liquid phase. The liquid phase can also comprise solid material functioning as growth and maintenance substrates for microorganisms to attach to.

The liquid phase may comprise carbon, nitrogen and/or phosphorous-comprising compounds, wherein carbon-comprising compounds may be formate or methanol, but preferably are understood to be essentially limited to dissolved CO, or urea, wherein the latter may also be considered a bioavailable Nsource. Formate or methanol can be converted to COin the liquid phase of the bioreactor, which can for example be catalysed by enzymes present inside or outside microorganisms, thereby indirectly acting as a supply of a gaseous substrate.

Gaseous phase is understood herein to mean a volume consisting of gaseous material and in contact with a liquid phase. The gaseous phase in a bioreactor is commonly called the headspace and is typically located directly above the liquid phase. To clarify, gas or gaseous substrate sparged into the liquid phase is not the gaseous phase, but becomes part of the gaseous phase upon exiting the liquid phase.

Gaseous substrate is understood to mean a gaseous supply of nutrients and/or energy for growth and/or maintenance of microorganisms.

Cement kiln is understood to mean a space used for the pyro-processing stage of manufacture of Portland and other types of hydraulic cement, in which calcium carbonate reacts with silica-bearing minerals to form a mixture of calcium silicates.

Syngas, or synthesis gas, is herein understood to mean a mixture comprising carbon monoxide, carbon dioxide and hydrogen. Syngas is produced by gasification of a carbon containing fuel to a gaseous product. The exact chemical composition of syngas varies based on the raw materials and the processes. One of the uses of syngas is as a fuel to manufacture steam or electricity. Another use is as a basic chemical building block for many petrochemical and refining processes. Syngas can be produced from many sources, including natural gas, coal, petroleum-based materials, biomass, other materials that would be rejected as waste, or virtually any hydrocarbon feedstock.

“Knallgas” is understood to mean a mixture of hydrogen and oxygen gases, which is highly combustible. A molar ratio of 2:1 is sufficient for maximum ignition efficiency.

Sparging is understood to mean a process in which a gas is bubbled through a liquid.

Dry weight or dry matter of a material herein is understood to mean the material consisting of all its constituents, but essentially excluding water. Examples of obtaining the dry weight or dry matter of a material are using centrifugation, drum drying, belt drying, evaporation, freeze drying, heating, spray drying, vacuum drying and/or vacuum filtration such that the water content of the material is removed.

Downstream processing is understood to mean one or more processing steps applied to a liquid phase removed from a bioreactor, which may include a kill-step process, a dewatering process, or a drying process.

Kill-step is understood to mean a process that achieves the reproductive inactivation of microorganisms. This process may occur within the liquid phase, a dewatered liquid phase, or dried biomass, for example by using an ultra-high-pressure homogeniser, acid, base, solvent, or heat-based microbial killing methods.

Dewatering is understood herein to mean a first process of removing liquid and/or a nutrient composition from biomass or a composition comprising biomass. Examples of dewatering are centrifugation, evaporation, heating, tangential flow filtration, and vacuum filtration. Dewatering may be followed by further downstream processing.

Drying is understood to mean a process of removing water from biomass or a composition comprising biomass to produce a biomass consisting of all its constituents, but essentially excluding water. Examples of drying are drum drying, belt drying, freeze drying, spray drying and vacuum drying.

Purifying carbon dioxide derived from the exhaust gas of a production or combustion process is understood to mean obtaining a volume consisting of essentially only carbon dioxide wherein other elements of the exhaust gas are substantially removed by devices and methods known in the art such as for example by using electrostatic precipitators or baghouses to remove ash and other particulates, using denitrification units to remove nitrogen oxides, using wet scrubbers, spray-dry scrubbers or dry sorbent injection systems to remove sulfur oxides. Carbon dioxide can be captured in post-combustion processes by separation methods known in the art, for example by using a solvent such as an amine to form a carbonate salt. The carbon dioxide is absorbed by the solvent after which it can be released by heat to form a highly purified stream of carbon dioxide.

Bioavailable nitrogen is understood to mean all nitrogen species that are readily available for uptake by microorganisms, including for example urea, ammonia, and amino-acids. For clarity, this is excluding dinitrogen (N).

Process limiting is understood to mean an instance where a substance can be measured as zero or close to zero in the liquid phase.

Chemostat is understood to mean a bioreactor in which the chemical environment is maintained in a more or less steady state with respect to for example microorganism concentration, pH, (dissolved) gaseous substrates, nutrient composition, liquid phase volume and other parameters known to a person skilled in the art.